Compound as cholinesterase inhibitor and its isolation from fungus sporotrichum species

ABSTRACT

The present invention provides a novel bioactive compound 12-(12′-CARBOXY-5′-METHOXYPHENYL)-2,12-DIHYDROXY-DODECA-4-ONE “Sporotricolone”, mainly as acetylcholinesterase (AchE) inhibitor, along with a process for the isolation of said compound from fungus Sporotrichum species.

FIELD OF INVENTION

The present invention relates to a compound12-(12′-CARBOXY-5′-METHOXYPHENYL)-2,12′-DIHYDROXY-DODECA-4-ONE“Sporotricolone”, mainly as acetylcholinesterase (AchE) inhibitor. Thepresent invention also relates to a process for the isolation of saidcompound from fungus Sporotrichum species.

BACKGROUND AND PRIOR ART REFERENCES

Enzyme inhibitors are important class of molecules that are used asdrugs and pesticides. The enzyme acetylcholinesterase (AchE) is involvedin the synaptic transmission of the nerve impulse and its inhibitionleads to accumulation of the neurotransmitter, acetylcholine leading tooverexcitation of the postsynaptic neuron. This property of theinhibitor has been exploited to develop newer insecticides against awide range of insect pests as well as drugs effective against worms,and, recently a new class of neuroactive drugs against dementia(Alzheimer's).

Although earlier authors have isolated metabolites such asasteric acis,questin and questinol from Sorotricum sp., no AchE inhibitor activityhas been reported (Slater, G P, Haskins, R H and Hogge, L. R. Can JMicrobiol 17 (1971), 1576-79). The fungi, Aspergillus terreus (Ling, KH, Liou, H H, Yang, C M and Yang C K, Appl. Env. Microbiol, 37 (1979)355-57) and Penicillium sp. (Omura, Skuno, F, Otoguro, K, Shiomi, K.Mauma, R and Iwai, Y. J. Antibiot. 48 (1995) 745-46) have been reportedto produce an AchE inhibitor named Arusigacin. However, the AchEinhibitor of the present invention is isolated from Sporotrichum havingdistinct chemical structure and properties and therefore a novelinhibitor molecule.

After screening of various microorganisms, a fungal culture is selectedwhich shows inhibition against a serine esterase/protease/cholinesteraseenzyme. This imperfect deuteromycetes, Sporotrichum species and wasfirst isolated in 1966. The taxonomic features of Sporotrichum species(deuteromyces) are broad hyphae and septae in nature; has hyallineconidiophores with little differentiation from vegetative hyphae andsolitary conidia with broad attachment to the hyphae.

This culture has previously been a subject of research investigation atthe Central Food Technological Research Institute (CFTRI) India, for itsability to grow on lignocellulosic wastes for the production of enzymesand organic acids (Sreekantaiah, K R, PhD thesis (1976) University ofMysore; Manonmani, H K, pHD thesis (1986) University of Mysore).

This culture has now been used in the present invention to produce afemented extract containing a serine esterase/protease/cholinesteraseinhibitor.

The conditions of fermentation have been described earlier in IndianPatent Application No. 303/DEL/2000 Sattur, A P, Shivanandappa, T,Divakar, S and Karanth, N G.

OBJECTS OF THE INVENTION

The main object of the present invention is to provide bioactivecompound 12-(2′-CARBOXY-5′-METHOXYPHENYL)-2,12-DIHYDROXY-DODECA-4-ONE“Sporotricolone” having inhibitory activity against acetylcholinesterase(AchE). Another object of the present invention is to provide a compoundhaving inhibitory activity against serine esterase and protease.

Yet another object of the present invention is to provide a compoundhaving insecticidal properties.

Still another object of the present invention is to provide a compoundhaving enhanced cholinergic activity.

Yet another object of the present invention is to provide a process forthe isolation of Sporotricolone.

SUMMARY OF THE INVENTION

The present invention provides a compound12-(2′-CARBOXY-5′-METHOXYPHENYL)-2,12-DIHYDROXY-DODECA-4-ONE“Sporotricolone” mainly as acetylcholinesterase (AchE) inhibitor. Thepresent invention also provides a process for the isolation of saidcompound from fungus Sporotrichum species.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, the present invention provides a bioactive compound12-(2′-CARBOXY-5′-METHOXYPHENYL)-2,12-DIHYDROXY-DODECA-4-ONE“Sporotricolone” of formula (I) having acetylcholinesterease (AchE)enzyme inhibition activity obtained from fungus Sporotrichum species.

An embodiment of the present invention wherein the compound12-(2′-CARBOXY-5′-METHOXYPHENYL)-2,12-DIHYDROXY-DODECA-4-ONE“Sporotricolone” of formula (I) having the following characteristicproperties:

Solubility: Highly soluble in ethyl acetate, methanol and acetone. UV(ethylene acetate) λ_(max): 265 nm, 312 nm.

¹HNMR spectrum (DMSO, δ_(TMS)=0.00 ppm); δ 1.03 (3H, d, J=6.3 Hz, —CH—CH₃) δ 1.2-2.7 (14H, m, 7x—CH ₂) δ 3.6-3.8 (m, Ar—O—CH ₃ and Ar—CH—OH) δ7.20 (1H, d, J=2.5 Hz, C_(6′)—H) δ 7.30 (2H, d, J=7.1 Hz, C_(3′)—H andC_(4′)—H)

Mass spectrum (EI, 70 eV, 25° C., 200-ul amp): m/e: 336 (M+),279(366-87), 167(274-112), 57(CH₂COCH₃), 43, 29.

Another embodiment of the present invention, wherein the purity of thecompound is established by TLC and RP HPLC.

Yet another embodiment of the present invention, wherein said compoundis named as12-(2′-CARBOXY-5′-METHOXYPHENYL)-2,12′-DIHYDROXY-DODECA-4-ONE“Sporotricolone”.

Still another embodiment of the present invention, wherein said compoundis an inhibitor of the enzyme acetylcholinesterase from the rat brain aswell as erythrocytes with a IC₅₀ value of 20×10⁻⁶ M.

Yet another embodiment of the present invention, wherein said compoundalso acts as an inhibitor of serine esterase of the rat liver serum.

Still another embodiment of the present invention, wherein said compoundhaving insecticidal properties.

Yet another embodiment of the present invention, wherein said compoundeffective against mosquito larvae at an optimum concentration of 70μg/ml water (70 ppm) when exposed for 24 hrs.

Still another embodiment of the present invention, wherein theinsecticidal activity of the compound against mosquito larvae isselected from culex quinquifasciatus.

Yet another embodiment of the present invention, wherein said compoundas acetylcholineesterase inhibitor having potential application as adrug for Alzheimer's disease or dermentia.

The present invention also provides a process for the isolation of12-(2′-CARBOXY-5′-METHOXYPHENYL)-2,12-DIHYDROXY-DODECA-4-ONESporotricolone from the fungus Sporotrichum species, said processcomprising the steps of:

(a) extracting the fermented solid with an organic solvent;

(b) filtering the extract of step (a) through a cloth or Whatman filterpaper to obtain a clear solution;

(c) evaporating the solution of step (b) under reduced pressure toobtain a crude extract;

(d) purifying the crude extract of step (c) by column chromatographyover silica gel and eluting with mixture of organic solvents ofincreasing polarity;

(e) pooling active eluted fraction of step (d) and further subjected tocolumn chromatography over silica gel by eluting with mixture of organicsolvents with increasing polarity;

(f) repooling the active eluted fractions of step (e);

(g) evaporating the pooled fractions of step (f) to get a residue; and

(h) dissolving the residue in step (g) in ethyl acetate to yield thepure compound “Sporotricolone”.

Yet another embodiment of the present invention, a process wherein instep (a) the organic solvent is selected from a group consisting ofethyl acetate, acetone or methanol and preferably ethyl acetate.

Still another embodiment of the present invention, a process wherein instep (d) the mixture of organic solvents is selected from thecombination of hexane: diethyl ether and chloroform:methanol mixtures.

Further embodiment of the present invention, a process wherein in step(e) the mixture of organic solvent used is chloroform:ethyl acetatemixture.

Yet another embodiment of the present invention, wherein the saidcompound is separated and purified by column chromatography on silicagel and RP HPLC.

Still another embodiment of the present invention, wherein said compoundhaving an UV absorption at 265 and 312 nm.

The present invention is further explained in the form of followingembodiments In the present invention a process for the isolation of anacetylcholinesterase inhibitor, which comprises the extraction of thefermented broth culture with solvents such as ethyl acetate. The crudeextract is further extracted with 10-20 ml of methanol and subjected tocolumn chromatography using silica gel and eluted with variouscombinations of solvents such as hexane:diethyl ether (85:15, 50:50)followed by chloroform:methanol (95:5, 50:50, 10:90). Fractions areevaporated under nitrogen, dissolved in ethyl acetate and assayed foracetylcholinesterase (AchE) inhibition. The active fractions are pooledand further subjected to purification on silica gel columnchromatography and eluted with chloroform:ethyl acetate (90:10, 50:50,0:100). The active fractions pooled and the solvent evaporated anddissolved in 2 ml ethyl acetate. The purity, as checked by TLC, showed asingle spot and HPLC on reverse phase column (C18) with chloroform andmethanol as mobile phase. The yield is about 10 mg. The purifiedinhibitor showed inhibitor potency against rat brain AchE with an IC₅₀of 15-20×10⁻⁶ M.

A general process for the production of the novel Acetylcholinesteraseinhibitor is given in the flow sheet.

12-(2′-CARBOXY-5′-METHOXYPHENYL)-2,12-DIHYDROXY-DODECA-4-ONE“Sporotricolone”

The structure of the isolated inhibitor is determined by UV, ¹H NMR andmass spectrometry.

The following examples are given by way of illustration of the presentinvention and should not be construed to limit the scope of theinvention.

EXAMPLE-1

The fermentation culture is extracted with 100 ml of ethyl acetate,filtered through a cotton filter and concentrated in vacuo to obtain 1ml of the crude extract. This is used as a source of the enzymeinhibitor and 20 μl of the extract gave 60-90% inhibition of rat brainacetylcholinesterase enzyme.

EXAMPLE-2

The crude extract is further extracted with 10-20 ml of methanol andsubjected to column chromatography using silica gel and eluted withvarious combinations of solvents such as hexane:diethyl ether (85:15,50:50) followed by chloroform:methanol (95:5, 50:50, 10:90). Fractionsare evaporated under nitrogen, dissolved in ethyl acetate and assayedfor acetyl cholinesterase (AchE) inhibitor. The active fractions(#11-19) are pooled and further subjected to purification on silica gelcolumn chromatography and eluted with chloroform:ethyl acetate (90:10,50:50, 0:100). The active fractions pooled and the solvent evaporatedand dissolved in 2 ml ethyl acetate. The purity, as checked by TLC,showed a single spot. RP HPLC also ascertained the purity on a C18column with chloroform and methanol as the mobile phase wherein it is asingle peak. The yield is about 10 mg.

EXAMPLE-3

The purified inhibitor showed inhibitor potency against rat brain AchEwith an IC50 of 15-20×10⁻⁶ M. as assayed according to Ellman et al.,(Biochem. Pharmacol. 7(1961), 88-95) and is given as follows: The enzymeinhibition is carried out by pre-incubating the enzyme (rat brainacetylcholinesterase) with 2-200 ul of the culture extract or the columnfraction at room temperature (25° C.) for 15 minutes followed by theaddition of the substrate, acetyl thiocholine iodide (0.5 mM), in 3 mlphosphate buffer (0.1 M, pH 7.4) containing 0.25 mMdithiobisnitrobenzoic acid. Absorbance change at 412 nm is monitoredevery 30 seconds for 2 min in an UV-VIS Spectrophotometer. Inhibition iscalculated relative to the solvent control. IC₅₀ is determined byregression analysis.

ADVANTAGES

1. The present invention provides an AchE/serine esterase/proteaseinhibitor from a microbial source.

2. The present invention provides a single extraction andchromatographic procedure to purify the AchE inhibitor from the crudemixture.

3. In the present invention the isolated inhibitor is a novel bioactivemolecule.

What is claimed is:
 1. A compound having the formula:


2. A method of killing or inhibiting the growth of an insect comprisingadministering to the insect an effective amount of the compound ofclaim
 1. 3. The method of claim 2 wherein said insect is a mosquito. 4.The method of claim 3 wherein said mosquito is Culex quinquifasciatus.5. A process for the isolation of the compound according to claim 1,said process comprising the steps of: (a) extracting fermented solidwith an organic solvent; (b) filtering the extract of step (a) through acloth or Whatman filter paper to obtain a clear solution; (c)evaporating the solution of step (b) under reduced pressure to obtain acrude extract; (d) purifying the crude extract of step (c) by columnchromatography over silica gel and eluting with mixture of organicsolvents of increasing polarity; (e) pooling active eluted fractions ofstep (d) and further subjected to column chromatography over silica gelby eluting with a mixture of organic solvents with increasing polarity;(f) repooling the active eluted fractions of step (e); (g) evaporatingthe pooled fractions of step (f) to yield the pure compound of formulaI; and (h) dissolving the residue in step (g) in ethyl acetate to yieldthe pure compound of claim
 1. 6. The process according to claim 5,wherein in step (a) the organic solvent is selected from the groupconsisting of ethyl acetate, acetone and methanol.
 7. The processaccording to claim 5, wherein in step (d) the mixture of organicsolvents is selected from the combination of hexane:diethyl ether andchloroform:methanol mixtures.
 8. The process according to claim 5,wherein in step (e) the mixture of organic solvent used ischloroform:ethyl acetate mixture.